Biotin (vitamin H) participates in cellular metabolism as the prosthetic group of carboxylases, decarboxylases, and transcarboxylases. The final step of biotin biosynthesis involves the insertion of sulfur into dithiobiotin, a chemically challenging reaction that entails the formation C-S bonds at inactivated, saturated carbon atoms. This transformation is dependent upon the bioB gene product, biotin synthase. Biotin synthase is an iron-sulfur (Fe-S) protein and belongs to the family of S-adenosylmethionine-dependent radical-generating Fe-S enzymes. New roles for Fe-S clusters are beginning to emerge in the generation of radical intermediates and in the donation of sulfur in the final step of biotin synthesis. Although the exact role of the Fe-S cluster is not fully understood, the use of the Fe-cluster as a sulfur donor in biotin biosynthesis implies novel S-based Fe-S cluster chemistry. Similar sulfur-based chemistry could be involved in the cleavage of S-adenosylmethionine to generate the 5?-deoxyadenosyl radical. The major objectives of this research proposal are to use molecular biology and biochemical methodologies together with the application of biophysical spectroscopic techniques (EPR, absorption/Cd/MCD, resonance Raman, and Mossbauer spectroscopies and mass spectrometry) to determine the mechanism of Fe-S cluster-mediated reductive cleavage of S-adenosylmethionine, the nature of the cluster transformation that occurs upon donation of the sulfur atom for biotin biosynthesis, and the requirements for catalytic biosynthesis of biotin.